Cells prepare for S-phase during G1 through the auspices of two AAA+ ATPases, ORC and CDC6, which function to load a latent helicase consisting of the MCM2-7 complex onto replication origins. The switch into S-phase is achieved by melting of the duplex origin by MCM2-7 and activation of the helicase by the addition the GINS and CDC45 accessory factors. The resultant CMG (Cdc45/Mcm2-7/GINS) assembly unwinds parental DNA strands and coordinates the synthetic enzymes of the replisome. Our long-term goal is to define the physical mechanisms by which MCM2-7 loading and CMG formation occur and are regulated in metazoans. Recent success in solving the first X-ray crystal structure of ORC suggests that the complex interconverts between inactive and active conformations. These results, along with other findings, have led us to develop a new model for ORC function in which the complex is recruited to origins and rendered competent to bind both DNA and CDC6 in an ATP-dependent manner in separate steps. In Aim 1, we will test aspects of this model and determine the path of duplex DNA in association with ORC and Cdc6 at molecular resolution. In Aim 2, we will look at a second key aspect of CMG formation, namely, how an essential co-loader (CDT1) associates with MCM2-7. Data from our lab and the literature suggest that, as with ORC, this step of replisome formation in metazoans is regulated by as yet undefined mechanisms. Finally, in Aim 3, we will examine key downstream events in the switch to S-phase, that is, how the CMG uses ATP to power processive DNA unwinding as an active DNA helicase, and how the CDC45 subunit of the CMG promotes stable MCM2-7?DNA interactions. Several significant findings are expected to arise from the proposed work; for example, understanding the mechanisms of MCM2-7 loading and CMG assembly will illuminate how the CMG physically functions to separate leading and lagging DNA strands prior to strand synthesis and how CMG accessory factor contribute to fork stability. At the same time, our new models for ORC and CDT1 regulation indicate that there exist as yet unidentified new pathways for regulating cell cycle progression, providing potentially novel targets and avenues for cancer therapy.